title: Crystallization and preliminary X-ray analysis of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27.
abstract: Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallographic studies. The crystals obtained belonged to the monoclinic space group P2(1), with unit-cell parameters a=39.52, b=70.68, c=95.42 Å, β=92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Gonçalves
authors.forename: Ana M
authors.initials: AM
authors.lastname: Esteves
authors.forename: Nuno
authors.initials: N
authors.lastname: Borges
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Matias
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D005988:Glyceric Acids / Q000378:metabolism; D008358:Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D055503:Protein Multimerization; D016963:Thermus thermophilus / Q000201:enzymology*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D005988:Glyceric Acids; C402688:mannosylglycerate; C005156:3-phosphoglycerate; D017853:Phosphotransferases (Alcohol Group Acceptor); C441640:mannosyl-3-phosphoglycerate phosphatase; D008358:Mannose